The present invention relates to the Schizochytrium genus SR21 strain and a microorganism belonging to the same species as does said SR21 strain or having substantially the same fungological properties as does said SR21 strain, the said SR21 strain and microorganism having the ability to produce a fat containing the (n-3) series of docosahexaenoic acid and the (n-6) series of docosapentaenoic acid, and the invention also relates to a process for preparing the (n-3) series of docosahexaenoic acid and/or the (n-6) series of docosapentaenoic acid utilizing said microorganisms.
In addition, the present invention relates to the fat produced by the above microorganisms, various feedstuffs and foods supplemented with the fat, as well as a process for utilizing the fat as an additive for various feedstuffs and foods.
It is believed that highly unsaturated fatty acids such as docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA), have a variety of physiological activities in animal bodies. It is known that these highly unsaturated fatty acids are divided into the (n-3) series and (n-6) series owing to the difference in the positions of their unsaturated bonds. In animal bodies, the (n-3) and (n-6) series of the highly unsaturated fatty acids belong to different metabolic pathways, and animals require both the acids as essential fatty acids.
The (n-3) series of highly unsaturated fatty acids include, for example, eicosapentaenoic acids [20:5 (n-3)], docosahexaenoic acid [22:6 (n-3)] or the like. It is known that these fatty acids have a physiological activity, such as anti-inflammatory or anti-thrombotic activity, and they are noted as a material for functional foods or for pharmaceuticals.
On the other hand, the (n-6) series of highly unsaturated fatty acids include, for example, xcex3-linolenic acids [18:3 (n-6)], di-homo-xcex3-linolenic acid [20:3 (n-6)], arachidonic acid [20:4 (n-6)] or the like, and they are noted as an intermediary metabolite toward the first or second group of eicosanoids, such as prostaglandins, leucotrienes or the like, which are referred to as local hormones.
In animal bodies, the final metabolite of the (n-3) series is docosahexaenoic acid and that of the (n-6) series is arachidonic acid, although they vary depending on tissues. For example, the fatty acid composition of phospholipids in human erythrocytes is as follows: for the (n-3) series, 0.70% eicosapentaenoic acid, 2.09% docosapentaenoic acid, 4.37% docosahexaenoic acid; on the other hand, for the (n-6) series, 12.67% linoleic acid, 0.62% di-homo-xcex3-linolenic acid, 16.93% arachidonic acid, 0.86% docosapentaenoic acid [Hardy et al., Biochem. J., vol. 274, p. 133 (1991)]. Thus, the amount of the (n-6) series of docosapentaenoic acid is very low.
The (n-3) series of docosahexaenoic acid (DHA) which is the final metabolite of the (n-3) series is present specifically in the brain or retina of animals, and it is believed that it performs some function in these organs. The (n-3) series of DHA is contained in oil from fish belonging to the group of blue fish, and in particular, contained in the oil from sardines or tuna in an amount of around 20%. Recently, due to the discovery of fish material containing a high concentration of DHA, such as the orbital fat of tuna, due to the progress of technology for purifying fatty acids well, and also due to other things, intensive efforts have been made to elucidate the physiological functions of DHA and to investigate its practical use. It has become apparent that DHA has physiological functions, such as an effect in lowering cholesterol, an anticoagulant effect and a carcinostatic effect. In relation to the brain metabolic system, it has also become apparent that DHA is effective for improving memory and learning ability, preventing senile dementia and treating Alzheimer""s disease. In addition, it has been proved that DHA is an essential fatty acid for the growth of fry. For reasons of the above, DHA is used as a material for health foods, baby milks or the like.
On the other hand, it is believed that the content of the (n-6) series of docosapentaenoic acid (DPA) in animal bodies increases in compensation for a lack of the (n-3) series of essential fatty acids. For example, in the fatty acid composition in optic nerve choroidea plexus of rats in the third generation after continuously receiving feed containing safflower oil which is very rich in the (n-6) series of fatty acids, the (n-3) series of DHA decreases to the degree of ⅓, while the (n-6) series of DPA increases 4 times [Homayoun et al., J. Neurochem., vol. 51, p. 45 (1988)]. Furthermore, it has been reported that the content of the (n-6) DPA in the hepatic microsome of rat having deficiency of vitamin A sharply increases from a normal value of 0.9% to 10.5% [Hamm et al., Biochem. J., vol. 245, p. 907 (1987)], that the (n-3) DHA decreases and the (n-6) DPA increases in rat receiving palm oil which is poor in components of the (n-3) series [Rebhung et al., Biosci. Biotech. Biochem., vol. 58, p. 314 (1994)], and so on.
Thus, the fact that the (n-6) DPA is produced in vivo in compensation for the (n-3) DHA which is suspected to perform a function in the brain or retina of animals suggests that the (n-6) DPA has a physiological role. In addition, the (n-6) DPA can be expected to be an antagonist to arachidonic acid.
Moreover, the following use of the (n-6) DPA is known at present: use of the same as a carrier for providing a tendency toward the easy transport of tranquilizer into the brain [Japanese Patent Publication (Kokai) No. 204136/1986] as well as use of the same in combination with the (n-6) series of docosatetraenoic acid for treating diseases in which the content of the (n-6) series of unsaturated fatty acids containing 22 carbon atoms is reduced from the normal level, for example, infection with a virus, in particular wart virus; leukemia, breast carcinoma and the other type of carcinomas; premenstrual syndrome and benign pectoral diseases; hypertension, hyperlipemia and obesity, dry eye syndrome; scleroderma, rheumatoid arthritis, Crohn disease, ulcerative colitis and the other type of autoimmune and inflammatory diseases; infertility; diabetes; psychotic diseases including schizophrenia and alcoholism (including influences of both excessive drinking and withdrawal effects) [Japanese Patent Publication (Kokai) No. 38324/1985].
The (n-6) DPA is not contained at all in fats usually supplied, but contained only in a small amount in fish oil together with the (n-3) DPA. Although a patent application directed to a process for isolating and/or concentrating the (n-6) DPA from the fish oil has been filed [Japanese Patent Publication (Kokai) No. 180849/1989], it is difficult to efficiently isolate and/or concentrate the same because the content of the (n-6) DPA in the fish oil is as little as 1%, the fish oil predominantly contains highly unsaturated fatty acids having a structure similar to that of the (n-6) DPA such as arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid, and the (n-3) DPA is contained in a content a few times higher than the (n-6) DPA, and therefore, the process requires, for example, a multistage chromatography treatment.
As described above, fish oil contains the (n-3) DHA and (n-6) DPA which have noteworthy physiological functions, but a fat containing these (n-3) DHA and (n-6) DPA in a large amount is not yet known. Furthermore, if one intends to use the fish oil, several disadvantages exist, such as the inability to maintain a stable source of fish oil due to the migration of fish or the like, or the offensive odor inherent in fish oil. Also, it is difficult to obtain a fat of reliable quality, because fish oil also contains highly unsaturated fatty acids, such as arachidonic acid (AA) and eicosapentaenoic acid (EPA), and is therefore easily oxidized. In addition, if one intends to obtain a highly purified (n-3) DHA or (n-6) DPA, it is difficult to separate and purify the same. In particular, when adding to a baby milk, it is desirable that the content of eicosapentaenoic acid is low, but it is very difficult to efficiently remove only eicosapentaenoic acid when using fish oil as the source.
Besides fish oil, fat accumulated in cultured cell bodies of a microorganism having the ability to produce the (n-3) series of docosahexaenoic acid is known as a source of the (n-3) series of docosahexaenoic acid. For example, the following microorganisms are known as those having the ability to produce the (n-3) series of docosahexaenoic acid: a bacterium separated from the deep sea, Vibrio marinus (ATCC 15381); Vibrio bacteria separated from the intestines of a deep-sea fish; micro-algae, Cyclotella cryptica and Crypthecodinium cohnii [Japanese Patent Publication (Kohyo) No. 503425/1993]; flagellate fungi, Thraustochytrium aureum (ATCC 34304) [Kendrick, Lipids, vol. 27, p. 15 (1992)] and Japonochytrium sp. (ATCC 28207) [Japanese Patent Publication (Kokai) No. 199588/1989]. According to a process utilizing these microorganisms, however, the amount of docosahexaenoic acid produced per liter (L) of medium is only on a very low level and in the order of several tens to 500 mg.
On the other hand, some of the micro-algae produce a fat containing docosapentaenoic acid, but the docosapentaenoic acid produced by each of these microorganisms is that of the (n-3) series. In addition, it is known that docosapentaenoic acid is also contained in flagellate fungi, Thraustochytrium aureum (ATCC 34304), Japonochytrium sp. (ATCC 28207) or the like, but it is reported that the acid produced by these microorganisms is also that of the (n-3) series. Thus, it was not previously known that the (n-6) series of docosapentaenoic acid is contained in the fat produced by these microorganisms in a sufficient amount.
The present inventors extensively sought a microorganism in marine microorganisms which produces much of a fat having a high content of the (n-3) series of docosahexaenoic acid (DHA) and/or the (n-6) series of docosapentaenoic acid (DPA) as mentioned above and having a low content of eicosapentaenoic acid (EPA).
As a result, they found that a certain kind of marine microorganism (assigned to a new species belonging to the Schizochytrium genus) produces much of a fat which contains not only the (n-3) DHA in a high content but also the (n-6) DPA and contains EPA in a low content, and thus they achieved the present invention.
Thus, the present invention provides the Schizochytrium genus SR21 strain and a microorganism belonging to the same species as does said SR21 strain or having substantially the same fungological properties as does said SR21 strain, the said SR21 strain and microorganism having the ability to produce a fat containing the (n-3) series of docosahexaenoic acid (DHA) and the (n-6) series of docosapentaenoic acid (DPA).
Also, the present invention provides a process for preparing a fat containing the (n-3) series of DHA and the (n-6) series of DPA, characterized in that the above microorganism is cultured and the above fat is recovered from the culture.
Furthermore, the present invention provides a process for preparing the (n-3) series of DHA or the (n-6) series of DPA, characterized in that it further encompasses a step of isolating the (n-3) series of DHA or the (n-6) series of DPA from the above fat.
In addition, the present inventors found that the above fat is useful as a source of the (n-3) DHA and/or (n-6) DPA for various feedstuffs and foods.
Thus, the present invention provides various feedstuffs and foods supplemented with the above fat.
Also, the present invention provides a process for utilizing the above fat as an additive for various feedstuffs and foods.